Abstract The broad goals of this research are to understand the mechanisms that promote stable, mitotically heritable states of gene expression during development. They continue to focus on characterizing the mutually antagonistic chromatin-directed activities of Drosophila Polycomb Group and Trithorax Group proteins that promote and maintain the distinct chromatin states associated with transcriptionally silent and active genes respectively. Their activities are implicated in many biological processes, including cell fate determination, stem cell maintenance and differentiation, regeneration, and others. They are implicated in human diseases, notably various cancers. The proposed experiments build on discoveries made in the previous funding period. Aim 1 is to investigate a newly discovered inhibitory activity of the Polycomb (PC) protein, a PRC1 subunit, on the catalytic activity of the CBP acetyltransferase, a global transcriptional co-activator. We will use CRISPR genome editing to create PC mutant proteins that fail to bind CBP in vitro and in vivo. We will characterize their effects in vivo on expression of both silent and active Polycomb-regulated genes, as well as on their genome- wide distribution. Aim 2 is to investigate the effects of newly discovered acetylation of PC on its in vivo function using CRISPR genome editing and transgenic lines that express PC containing substitutions of acetylated lysines with glutamines (acetyl-K mimic) and arginines (charge conservative but unacetylatable). Aim 3 is to investigate the interplay of the chromatin-directed activities of TRX methyltransferase and CBP acetyltransferase, extending our observation that the histone H3K4me1 product of TRX stimulates its subsequent acetylation of H3K27 by CBP, which directly blocks repressive H3K27 methylation by PRC2. We will determine if binding of CBP to TRX is required for this effect in vivo by mutating the CBP-binding region of TRX. This work will provide new insights into the regulation of active and silent chromatin states during development. .